This proposal is designed to yield new insights into an important feature of protein folding, the process of disulfide bond formation, and to provide biotechnological tools for enhanced production of multidisulfide proteins. I. We will study the process of disulfide bond isomerization in the Escherichia coli periplasm in the following ways: a) We will determine the pathways of electrons from cytoplasm to periplasm that insure the maintenance of disulfide bond isomerases in the reduced state; b)We will determine the specificity of the disulfide bond isomerases, DsbC and DsbG, and of the protein necessary for their reduction, DsbD; c)We will alter the specificity and activity of these proteins using mutagenesis and gene scrambling in order both to understand the mechanism of their action and to enhance the production of heterologous disulfide-bonded proteins; d)We will characterize new genes, which in multi-copy or when altered by mutation, enhance the production of multi-disulfide proteins in the periplasm. II. We will also study the process of disulfide bond formation and isomerization in the cytoplasm of strains of E. coli with altered thiol:disulfide redox environments. Beginning with strains missing the two major thiol: disulfide redox pathways, we have isolated suppressor mutations that restore sufficient reductive power to the cytoplasm for growth, but still allow cytoplasmic formation of protein disulfide bonds. Studies of these suppressors are already revealing novel interactions among the large set of cytoplasmic redox proteins. We will determine the altered genes and the mechanisms whereby suppressor mutations generate the paradoxical reducing but oxidizing cytoplasms. We will analyze suppressor for the kinetics of disulfide bond formation and isomerization and for the efficiency in the production of heterologous disulfide bonded proteins. This proposal thus unites extensive basic studies on processes of electron transfer in thiol: redox reactions with likely benefits for the biotechnological production of medically useful products.